General Metabolism III: Cofactors and Common Themes Andy Howard Introductory Biochemistry 26 November 2013 Biochemistry: Metabolism III 11/26/2013

Common themes in intermediary metabolism Many of the metabolic systems on which you’ll focus in the spring have common themes uniting them. 11/26/2013 Biochemistry: Metabolism III

Metabolism topics Specific systems Cosubstrates Prosthetic groups SAM, UDP-glucose Coenzyme A NAD(P) Prosthetic groups Flavins TPP, PLP Biotin, THF Cobalamin, lipoamide Common themes Specific systems Glycolysis and gluconeogenesis TCA cycle and electron transport Photosynthesis Lipid metabolism Amino acid metabolism Nucleic Acid metabolism 11/26/2013 Biochemistry: Metabolism III

S-adenosylmethionine Made from methionine and adenosine Sulfonium group is highly reactive: can donate methyl groups S-adenosyl-methionine methionine ATP Pi+PPi This sulfur is chiral; only one enantiomer is active! 11/26/2013 Biochemistry: Metabolism III

UDP-glucose Most common donor of glucose Formed via: Glucose-1P + UTPUDP-glucose + PPi Reaction driven to right by PPi hydrolysis UDP-glucose 11/26/2013 Biochemistry: Metabolism III

Thioesters: another class of high-energy compounds Thioesters have similar reactivity as oxygen-acid anhydrides Thioesters less stable than oxygen esters because the unshared electrons in sulfur are not as delocalized in a thioester as the unshared electrons in an oxygen ester 11/26/2013 Biochemistry: Metabolism III

Coenzyme A (G&G p.616) Reactive portion is free sulfhydryl at one end of the molecule Can form thioester with acetate, etc. Pantoate + b-alanine = pantothenate 2-mercapto-ethylamine) -alanine) (ADP-3’P) (Pantoate) 11/26/2013 Biochemistry: Metabolism III

NAD+ and NADP+ Net charge isn’t really > 0 ; the + is just a reminder that the nicotinamide ring is positively charged Most important cosubstrates in oxidation-reduction reactions in aerobic organisms Nicotinamide adenine dinucleotide phosphate 11/26/2013 Biochemistry: Metabolism III

Differences between them The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety The functional difference is that NAD+ is usually associated with catabolic reactions and NADP+ is usually associated with anabolic reactions Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products Exceptions: photosynthesis and ETC! 11/26/2013 Biochemistry: Metabolism III

How do we get back to the starting point? NADH is often oxidized back to NAD+ as part of the electron-transport chain NADPH is created via photosynthesis Imbalances can be addressed via NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase 11/26/2013 Biochemistry: Metabolism III

Reduced forms of NAD(P) Reduction occurs on the nicotinamide ring Ring is no longer net-positive Ring is still planar but the two hydrogens on the para carbon are not 11/26/2013 Biochemistry: Metabolism III

NADPH Provides reducing power for anabolic reactions Often converting highly oxidized sugar precursors into more reduced molecules 11/26/2013 Biochemistry: Metabolism III

FAD and FMN (p. 615) Flavin group based on riboflavin Flavin mononucleotide Flavin group based on riboflavin Alternate participants in redox reactions Prosthetic groups: tightly but noncovalently bound to their enzymes That protects against wasteful reoxidation of reduced forms FADH2 is weaker reducing agent than NADH These are capable of one-electron oxidations and reductions 11/26/2013 Biochemistry: Metabolism III

Flavin adenine dinucleotide (FAD) (adenosine monophosphate) FAD and FMN structures Flavin adenine dinucleotide (FAD) FAD has an AMP attached P to P (riboflavin) 11/26/2013 Biochemistry: Metabolism III

FMN/FAD redox forms Two-electron version: H+ + :H- transferred Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University 11/26/2013 Biochemistry: Metabolism III

Thiamine Pyrophosphate (G&G p. 614) Based on thiamine, vitamin B1 Carboxylases and oxidative decarboxylases use this coenzyme So do transketolases (move 2 carbons at a time between sugars with keto groups) Thiazolium ring is reactive center: pKa drops from 15 in H2O to 6 in enzyme 11/26/2013 Biochemistry: Metabolism III

TPP Ylid form of TPP We already talked about decarboxylations of -ketoacids, e.g. pyruvate + H+  acetaldehyde + CO2 Formation and cleavage of -hydroxylactones & -hydroxyacids: 2 pyruvate + H+  acetolactate + CO2 acetolactate 11/26/2013 Biochemistry: Metabolism III

TPP reactions pyrimidine thiazolium Diagram courtesy of Oklahoma State U. Biochemistry program 11/26/2013 Biochemistry: Metabolism III

Pyridoxal phosphate PLP is prosthetic group for many amino-acid-related enzymes, including, but not limited to, transaminations That’s how a lot of -amino acids are synthesized from the corresponding -ketoacids: α-keto acid 1 α-amino acid 2 α-keto acid 2 α-amino acid 1 11/26/2013 Biochemistry: Metabolism III

How PLP functions Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine 11/26/2013 Biochemistry: Metabolism III

PLP Remember we said it gets used in a lot of transaminations We should consider its chemistry and its other roles in pathways To start with: it exists in at least 2 tautomeric forms 11/26/2013 Biochemistry: Metabolism III

PLP: Non-transamination reactions -decarboxylation: -amino acid + H+  CO2 + H3N+-CH2-R -decarboxylation 11/26/2013 Biochemistry: Metabolism III

Biotin (cf. fig. 22.3) Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl groups … and in ATP-dependent carboxylations biotin 11/26/2013 Biochemistry: Metabolism III

Biotin reactivity Covalently bound to active-site lysines to form species called biocytin Pyruvate carboxylase is characteristic reaction: Diagram courtesy University of Virginia Biochemistry 11/26/2013 Biochemistry: Metabolism III

Tetrahydrofolate (G&G pp. 894-895) Primary donor of one-carbon units (formyl, methylene, methyl) Supplies methyl group for thymidylate Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells Trimethoprim as antibacterial: Bacterial DHFR is somewhat different from eukaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate 11/26/2013 Biochemistry: Metabolism III

THF structure and function Figure courtesy horticulture program, Purdue 11/26/2013 Biochemistry: Metabolism III

Tetrahydrofolate variations -2 oxidation state: methyl donor from N5-methyl-THF 0 oxidation state: methylene donor from N5,N10-methylene-THF +2 oxidation state: formyl (-CH=O) from N5-formyl-THF and N10-formyl-THF Formimino (-CH=NH) from N5-formimino-THF Methenyl (-CH=) from N5,N10-methenyl-THF See textbook for specifics 11/26/2013 Biochemistry: Metabolism III

Cobalamin Largest B vitamin Structure related to heme but missing one carbon in ring structure Cobalt bound in core of ring system Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation 11/26/2013 Biochemistry: Metabolism III

Adenosyl- Cobalamin Reactive Co-C bond “Missing” carbon Diagram courtesy of Swiss Food News 11/26/2013 Biochemistry: Metabolism III

Lipoamide (p.616) Protein-bound form of lipoic acid enzyme Lipoamide (p.616) lipoate Protein-bound form of lipoic acid Contains five-membered disulfide ring Covalently bound via amide to protein lysine sidechain Involved in swinging arm between active sites in multienzyme complexes Disulfides break periodically Example: pyruvate dehydrogenase complex 11/26/2013 Biochemistry: Metabolism III

Lipoamide 2e- reduction thioester starting point 11/26/2013 Biochemistry: Metabolism III

iClicker quiz question 1 Based on what you have learned, would you expect glycogen synthase to be activated or inhibited by phosphorylation? (a) activated (b) inhibited (c) neither (d) insufficient information to tell 11/26/2013 Biochemistry: Metabolism III

iClicker quiz question 2 What would you expect to be the phosphate donor in the NAD kinase reaction? (a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate 11/26/2013 Biochemistry: Metabolism III

iClicker quiz question 3 Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate  aspartate + a-ketoglutarate? (a) ascorbate (b) PLP (c) thiamine pyrophosphate (d) NAD (e) none of the above 11/26/2013 Biochemistry: Metabolism III

Common themes & specifics Every major biochemical pathway: Involves enzymatic activity Employs cofactors Includes oxidation-reduction reactions Contributes to cellular homeostasis Is subject to control via thermodynamics, localization, transcription, and allostery We’ll explore the specifics a little now 11/26/2013 Biochemistry: Metabolism III

Glycolysis & gluconeogenesis Glycolysis: Conversion of sugars (glycogen, glucose, glucose-6-P, fructose, etc.) to pyruvate (catabolic) Gluconeogenesis: buildup of pyruvate and TCA cycle intermediates to make glucose or glucose-6-P (anabolic) 7 common reactions, 3 divergences due to irreversibility 11/26/2013 Biochemistry: Metabolism III

Glycolysis (table 18.1) Reactants Products Enzyme ΔGo’ ΔG Glucose,ATP G6P,ADP Hexokinase -16.7 -33.9 G6P F6P Phosphoglucoisomerase 1.7 -2.9 F6P+ATP F1,6bisP+ADP Phosphofructokinase -14.2 -18.8 F-1,6bisP DHAP,Glyc3P Aldolase 23.9 -0.23 DHAP Glyc3P Triosephosphate isomerase 7.6 2.4 Glyc3P,NAD,Pi 1,3bisPgl,NADH Glyc-3-P dehydrogenase 6.3 -1.3 1,3bisPgl,ADP 3-P-glcate,ATP Phosphoglycerate kinase -18.9 0.1 3-P-glcate 2-P-glycate Phosphoglycerate mutase 4.4 0.8 2-P-glcate+H2O PEP Enolase 1.8 1.1 PEP+ADP Pyruvate +ATP Pyruvate kinase -25.2 -14.8 11/26/2013 Biochemistry: Metabolism III

Gluconeogenesis (§§22.1-22.2) Reverses all 7 of the reactions in the previous slide that have ΔG > -5 Differences: Reactants Products Enzyme ΔGo’ ΔG Pyruvate+ATP+CO2 Oxalo-acetate Pyruvate carboxylase -12? -4? OAA PEP PEP carboxykinase +12? ~ 0 F1,6bisP F6P Fructose 1,6-bisphosphatase ~ -18 ~ -22 G6P Glucose Glucose 6-phosphatase ~ -15 11/26/2013 Biochemistry: Metabolism III

TCA cycle (Chapter 19) Pyruvate has a variety of fates, one of which is to be decarboxylated and oxidized to acetyl CoA via pyruvate dehydrogenase The acetyl CoA then can condense with oxaloacetate to form a tricarboxylic acid, citrate Citrate then goes through a series of oxidative transformations that yield 3 NADH, 1 FADH2, and one GTP as it gets converted to oxaloacetate, which can re-enter the cycle 11/26/2013 Biochemistry: Metabolism III

Electron transport (Chapter 20) The NADH and FADH2 produced in the TCA cycle (or elsewhere) can be reoxidized to NAD and FAD, using O2 as the ultimate electron acceptor Every NADH reoxidized yields 2.5 ATP Every FADH2 reoxidized yields 1.5 ATP The ATP production occurs via a proton pump mechanism in ATP synthase 11/26/2013 Biochemistry: Metabolism III

Other sugar pathways (§§22.3-22.6) Pentose phosphate pathway yields NADPH (which anabolic steps require) and interconverts various sugar phosphates Glycogen and starch are sugar storage molecules; they can be made and unmade Glyoxalate pathway (absent in animals) short-circuits the TCA cycle; allows acetyl CoA to serve as a source of sugars 11/26/2013 Biochemistry: Metabolism III

Plant biochemistry (Chapter 21) Plants, bluegreens, some other organisms can capture light energy and use it to ionize molecules or convert electrons to excited states: photosynthesis Most of these can grab atmospheric carbon dioxide and incorporate its carbon into organic molecules: RuBisCO Various interconversions (Calvin cycle) build sugars from there. 11/26/2013 Biochemistry: Metabolism III

Lipid metabolism (Chapters 23 and 24) Fatty acids are lengthened, 2 C at a time, via malonyl ACP condensing with a growing chain, up to 16 or 18 C, in a single enzymatic complex Fatty acids are broken down, 2C at a time, via acetyl CoA units Isoprenoids are built up from mevalonate Lipid molecules are built up from, and broken down into, FAs and isoprenoid units 11/26/2013 Biochemistry: Metabolism III

Amino acid anabolism (Chapter 25, esp. §25.4) Every amino acid has its own pathway Many involve transaminations α-ketoacid1 + α-amino acid2  α-aminoacid1 + α-ketoacid2 Most of the precursors are derived from TCA cycle intermediates, with or without phosphorylation 11/26/2013 Biochemistry: Metabolism III

Amino acid catabolism (§25.5) Again, every story is slightly different Many of the products lead pyruvate or TCA cycle intermediates; these are considered glucogenic Some lead to acetyl CoA or acetoacetate and are considered ketogenic; only lys and leu are purely ketogenic. 11/26/2013 Biochemistry: Metabolism III

Nucleic acids (Chapter 26) Nucleosides synthesized from glutamine, aspartate, glycine, phosphoribosyl pyrophosphate Interconversions are well-understood Salvage pathways are surprisingly (to me, at least) important medically 11/26/2013 Biochemistry: Metabolism III

Molecular biology as a biochemical subject (Chapters 28 and 29) DNA replication, transcription, and translation are proper subjects for biochemical analysis Enzymes are well-defined and carefully regulated Some of the enzymes are RNA rather than proteins 11/26/2013 Biochemistry: Metabolism III